Approaches are being explored to increase the therapeutic efficacy of the fluorinated pyrimidine, 5-fluorouracil (5-FU), by the modulation of its intracellular metabolism. Recent experimental work has initiated various clinical trials directed at enhancing the incorporation of 5-FU into tumor cell RNA through combinations with thymidine (TdR) and N-phosphonacetyl-L-aspartate (PALA). We are currently conducting a phase II study of combination therapy with PALA and 5-FU. Preliminary studies have indicated the feasibility of monitoring 5-FU incorporation into RNA isolated from bone marrow, oral mucosa and tumor biopsies. The application of this methodology provides a basis from which the formation of (5-FU)RNA can be correlated with both toxicity and antitumor effect. We have also been exploring the effects of combination therapy with TdR, PALA, methotrexate (MTX) and 6-methyl-mercaptopurine riboside (MMPR) on human tumor cells in culture with respect to the formation of phosphoribosyl-1-pyrophosphate (PRPP) and the enhancement of 5-FU incorporation into RNA. Drug combinations have been employed that increase the synthesis of PRPP by 15-20 fold and result in a similar enhancement of (5-FU)RNA formation. The objective of this proposal is to develop more effective therapeutic approaches through the modulation of 5-FU metabolism. We propose to: (1) continue ongoing studies monitoring the incorporation of 5-FU into RNA in specimens from patients receiving PALA and 5-FU in a phase II study, (2) extend human tissue culture studies exploring the enhancement of (5-FU)RNA formation using combinations of TdR, PALA, MTX and MMPR and to further investigate these drug combinations in an animal tumor model to monitor (5-FU)RNA formation in vivo as a correlate with antitumor response, and (3) extend the phase II PALA/5-FU based upon tissue culture and animal data by the addition of agents to enhance the formation of PRPP as a correlate of increased 5-FU incorporation into tumor RNA. These studies are important in terms of providing a rational basis for the guidance of future clinical trials with 5-FU.